Primers used in this study.

Vibrio species are marine prokaryotes that inhabit diverse ecological niches, colonizing abiotic and biotic surfaces. These bacteria are vital players in the global carbon cycle, assimilating billions of tonnes of chitin for carbon (and nitrogen) metabolites. Many bacterial proteins involved in the process-including chitinases, sugar transporters, and modifying enzymes-have been well studied. However, the genetic functional interplay and key drivers of Vibrio competitive survival in the presence of chitin as the dominant carbon source is not understood. To address this question, we carried out transposon sequencing (Tn-seq) to determine the genetic fitness of Vibrio parahaemolyticus mutants grown on chitin as a sole carbon source. Along with validating known Vibrio genes associated with chitin metabolism, our data newly identified vital roles for an unclassified OprD-like import chitoporin and a HexR family transcriptional regulator. Furthermore, we functionally implicated HexR in regulating multiple physiological processes involved in V. parahaemolyticus environmental survival including carbon assimilation and cell growth, biofilm formation, and cell motility. Under nutrient limiting conditions, our data revealed a requirement for HexR in filamentous cell morphology, a critical trait for V. parahaemolyticus environmental fitness. Therefore, a vital import porin and genomic regulation mediated by HexR support multiple physiological processes for Vibrio chitinolytic growth and environmental fitness.